Steroidogenic factor 1 (SF-1) (also called Ad4BP or NR5A1) is a member of the nuclear receptor superfamily, which consists of DNA binding transcription factors (39). In contrast to other nuclear receptors, which are activated by ligands (51), nuclear receptors 5A are orphan receptors because the existence of their ligands is still under debate (31, 53). One characteristic of the nuclear receptor 5A family is the presence in all family members of a conserved region called the Ftz-F1 box that functions in the recognition of a DNA sequence (52).

Unresectable hepatic colorectal cancer (CRC) metastases are a leading cause of cancer mortality. These tumors and other epithelial tumors often express both cyclooxygenase-2 (COX-2) and carcinoembryonic antigen (CEA). Because adenovirus (Ad) vectors infect the liver and lack tumor tropism, they cannot be used for systemic therapy of hepatic metastases. We used COX-2 transcriptional restriction, in combination with transductional Ad hepatic untargeting and tumor retargeting by a bispecific adapter, sCARhMFE, composed of sCAR [the coxsackie/Ad receptor (CAR) ectodomain] and MFE-23 (a single-chain anti-CEA antibody), to untarget liver after i.v. administration of Ad vectors expressing firefly luciferase and to retarget virus to hepatic colorectal tumor xenografts and non-small cell lung tumor xenografts. To improve both liver untargeting and tumor retargeting, we developed sCARfMFE, a trimerized sCARhMFE adapter. Trimerization greatly improves both untargeting of CAR-dependent Ad infection and CEA-dependent virus retargeting in culture and in vivo. Combining sCARfMFE bispecific adapter transductional liver untargeting and transductional tumor retargeting with COX-2 transcriptional tumor-restricted transgene expression increases systemically administered Ad therapeutic efficacy for hepatic CRC tumors, using herpes virus type 1 thymidine kinase (HSV1-tk) as a therapeutic gene in conjunction with the prodrug ganciclovir (GCV). Both transductional untargeting and COX-2 transcriptional restriction also reduce HSV1-tk/GCV hepatic toxicity. In addition, transductional sCARfMFE untargeting reduces the innate immune response to systemic Ad administration. Combined transductional liver Ad untargeting, transductional tumor retargeting, and transcriptional transgene restriction suggests a means to engineer practical, effective therapeutic agents for hepatic CRC metastases in particular, as well as hepatic metastases of other epithelial cancers. [Cancer Res 2009;69(2):554-64]

Mesenchymal stem cells (MSCs) have been used in clinical studies to treat neurological diseases and damage. However, implanted MSCs do not achieve their regenerative effects by differentiating into and replacing neural cells. Instead, MSC secretome components mediate the regenerative effects of MSCs. MSC‐derived extracellular vesicles (EVs)/exosomes carry cargo responsible for rescuing brain damage. We previously showed that EP4 antagonist‐induced MSC EVs/exosomes have enhanced regenerative potential to rescue hippocampal damage, compared with EVs/exosomes from untreated MSCs. Here we show that EP4 antagonist‐induced MSC EVs/exosomes promote neurosphere formation in vitro and increase neurogenesis and neuritogenesis in damaged hippocampi; basal MSC EVs/exosomes do not contribute to these regenerative effects. 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNP) levels in EP4 antagonist‐induced MSC EVs/exosomes are 20‐fold higher than CNP levels in basal MSC EVs/exosomes. Decreasing elevated exosomal CNP levels in EP4 antagonist‐induced MSC EVs/exosomes reduced the efficacy of these EVs/exosomes in promoting β3‐tubulin polymerization and in converting toxic 2′,3′‐cAMP into neuroprotective adenosine. CNP‐depleted EP4 antagonist‐induced MSC EVs/exosomes lost the ability to promote neurogenesis and neuritogenesis in damaged hippocampi. Systemic administration of EV/exosomes from EP4‐antagonist derived MSC EVs/exosomes repaired cognition, learning, and memory deficiencies in mice caused by hippocampal damage. In contrast, CNP‐depleted EP4 antagonist‐induced MSC EVs/exosomes failed to repair this damage. Exosomal CNP contributes to the ability of EP4 antagonist‐elicited MSC EVs/exosomes to promote neurogenesis and neuritogenesis in damaged hippocampi and recovery of cognition, memory, and learning. This experimental approach should be generally applicable to identifying the role of EV/exosomal components in eliciting a variety of biological responses.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAM/CD49f luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAM/CD49f basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAM/CD49f luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAM/CD49f luminal mammary epithelial cells.